Project Summary The cytoskeleton and its connections to the nucleus play fundamental roles in establishing cellular morphology, polarity, migration and adhesion. Because of their essential roles in these cellular functions, it is critically important to understand the changes that take place in cytoskeletal systems and their nuclear connections during the normal process of cellular aging. Despite this importance, remarkably little is known about the role of the cytoskeleton in physiologically aging. Our preliminary studies have led to the discovery of a fundamental cell polarity defect that occurs in fibroblasts from children with the accelerated aging disorder Hutchinson- Gilford progeria syndrome and also in fibroblasts from physiologically-aged individuals. This defect results from unbalanced connections between the nuclear lamina on the inner aspect of the inner nuclear membrane and two major cytoskeletal protein systems: actin microfilaments and microtubules. These connections are mediated by the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex composed of inner nuclear membrane SUN and outer nuclear membrane KASH/nesprin proteins. In aging, there is a preferential increased interaction of nesprin-2G with microtubules (supported by SUN1) versus actin microfilaments (supported by SUN2). This has led to our overall hypothesis that altered nucleocytoskeletal connections mediated by the LINC complex causes an intrinsic cell polarity defect in physiological aging as well as an accelerated aging disorder. We further hypothesize that this defect is at least in part mediated by a protein factor secreted by cells from aged individuals. We propose to test these hypotheses in three specific aims. In Aim 1, we will decipher the mechanism underlying the cell polarity defect in physiological aging. We will determine how increased SUN1 levels are established in the nuclear envelope during aging, explore how SUN1 interaction with nesprin-2G biases its interaction toward microtubules and examine how cytoplasmic microtubules are impacted by their excessive interaction with the nuclear envelope. In Aim 2, we will examine the role of the age-dependent cell polarity defect in would healing in vivo. We will examine cutaneous wounds from young and old mice to determine if the same mechanisms that generate polarity defects in tissue culture are operative in vivo as well as examine wound healing and fibroblast polarity in transgenic mice overexpressing SUN1 in fibroblasts. In Aim 3, we will purify and characterize a soluble factor secreted from aged cells that leads to defective cell polarity. We will use complementary methods of mass spectrometry and protein purification to identify this factor we call FRAC, for Factor Released from Aged Cells. Completion of these aims will lead to novel insights into how alterations in nucleocytoskeltal connections at the LINC complex lead to a fundamental cell polarity defect in aging.